Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Two new iridoid glucosides from Ixora chinensis

From leaves and twigs of Ixora chinensis, two new iridoid glucosides, ixoroside (1) and ixoside (7,8-dehydroforsythide) (2) along with known geniposidic acid (3) have been isolated and their structures have been established.     

Procedure for the efficient acquisition of progeny seeds from crossed potato plants grafted onto tomato

Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.     

Solaniol, a Toxic Metabolite of Fusarium solani

Fusarium solani M-1-1 isolated from moldy bean hulls produces T-2 toxin, diacetoxyscirpenol, and a new toxic trichothecene, solaniol, in Czapek-Dox-peptone medium.     

Cooperative conformational change and aggregation of concanavalin A in alkaline solutions

Three properties, the binding activity to Sephadex G-75, conformation, and the extent of aggregation, of concanvalin A. (con A) in alkaline pH solutions were examined with special attention to the time course and their time-independent final values. Highly cooperative conformational changes among four subunits were suggested which were coupled either with protonation in the case of demetallized con A or with metal binding in the case of metal-liganded con A. Midpoints of the conversions of the metal-liganded con A were about pH 8.8, 9.1 and 9.1 with respect to the activity, the conformational change and the aggregation, respectively. These values were about 1 pH higher than the corresponding values of demetallized con A: 7.9, 8.05 and 8.2. Each conversion took place in narrow pH ranges. The pH range for the loss of activity was found to be significantly lower than those of the other two. The aggregation was suggested not to be coupled with the conformational change. Dissociation into subunits did not take place indicating strong interactions among four subunits in the tetramer.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Secretory type of recombinant thioredoxin h induces ER stress in endosperm cells of transgenic rice

Thioredoxin h (TRX h) functions as a reducing protein and is present in all organisms. As a new approach for inducing the endoplasmic reticulum (ER) stress, TRX h (OsTRX23) was expressed as a secretory protein using the endosperm-specific glutelin GluB-1 promoter and a signal peptide. In transgenic rice seeds, the majority of the recombinant TRX h accumulated in the ER but some was also localized to the protein body IIs (PB-IIs). The rice grain quality was dependent on the TRX h accumulation level. Increased TRX h expression resulted in aberrant phenotypes, such as chalky and shriveled features, lower seed weight and lower seed protein content. Furthermore, the accumulation of some seed storage proteins (SSPs) was significantly suppressed and the morphology of the protein bodies (PB-Is and PB-IIs) changed according to the level of TRX h. SSPs, such as 13 kDa prolamin and GluA, were specifically modified via the reducing action of TRX h. These changes led to the activation of the ER stress response, which was accompanied by the expression of several chaperone proteins. Specifically, the ER stress markers BiP4 and BiP5 were significantly up-regulated by an increase in the level of TRX h. These results suggest that changes in the conformation of certain SSPs via the action of recombinant TRX h lead to an induced ER stress response in transgenic rice seeds.